This invention relates to the separation and detection of spermatozoa. It provides methods and kits for the separation and/or detection of motile spermatozoa in a sample, which are useful in a number of applications, including the diagnosis and treatment of male infertility.
It has been estimated that approximately 14-16% of all couples attempting to conceive experience difficulty, and are defined by fertility therapists as infertile. 40% of these cases result from male factors. In a substantial proportion of these, treatment is available to ameliorate or relieve the condition which leads to infertility.
Other conditions also exist in which it is desirable to test for the presence or otherwise of viable spermatozoa in a sample. For example, vasectomies are now frequently carried out as a method of contraception, but it is necessary to verify the effectiveness of a vasectomy by confirming that ejaculate is free of viable spermatozoa for a period of time after the operation.
A number of methods exist for assessing the motility and number of spermatozoa in a sample. One such method is microscopic analysis, which is typically carried out in a hospital or commercial laboratory. More recently, however, a number of proposals have been made for test kits which are intended to simplify the detection of spermatozoa, and which may therefore be useful in the diagnosis of male infertility. For example, WO97/40386 discloses a kit which is based on the detection of the 34 kD human epididymal spermatozoa protein (P34H). This protein is thought to be involved in spermatozoa-zona pellucida interaction. The test kit disclosed in WO97/40386 uses an antibody raised against P34H or a related antigen, and a reagent for detecting antibody binding to P34H. As disclosed in WO97/40386, spermatozoa in a test sample are washed three times by centrifugation in Dulbecco-phosphate buffered saline. The samples are then heat denatured at 95xc2x0 C., centrifuged at 14000 g, and the supernatants are then used for analysis.
EP-A-0387873 also discloses a kit for the evaluation of male fertility. This kit uses solid beads to which is bound an antibody specific to an antigenic site on the human spermatozoon acrosome. Such beads are mixed with a test sample, and incubated for a period of 10 to 30 minutes. The test beads are then separated from the suspension, washed and subjected to measurement of the number of spermatozoa bound to the solid beads, preferably by examination with the aid of a microscope.
A kit for the detection of spermatozoa in a sample is also disclosed in WO95/29188. In this case, the test is based on antibodies to an antigen such as the SP-10 antigen of human spermatozoa.
A significant disadvantage of the test kits disclosed in the prior art mentioned above is that they do not distinguish between motile and non-motile spermatozoa. In the detection of male infertility, the ability to assess the numbers of motile spermatozoa is the most predictive indicator of male infertility. Moreover, many of the prior art test kits involve procedures, such as centrifugation or microscopic examination, which do not lend themselves to home use, instead requiring implementation by a skilled practitioner.
It is therefore desired to provide a device, which can be self contained or provided in a plurality of components, and a method for separating motile spermatozoa from non-motile spermatozoa, and for collecting and detecting the presence of the motile spermatozoa.
The invention provides an apparatus for separating motile spermatozoa from non-motile spermatozoa in a liquid sample, the apparatus comprising (i) a vessel having a sample receiving inlet, a filtered sample outlet and a sample separation filter mounted therebetween, the sample separation filter having a sample-receiving surface and an opposed surface, and the sample separation filter being effective substantially to prevent flow of the sample therethrough, but permitting passage of motile spermatozoa therethrough when said opposed surface of said sample separation filter is placed in contact with a liquid medium and (ii) means for supplying a liquid to said opposed surface of said filter. The sample may comprise motile spermatozoa, non-motile spermatozoa and/or spermatozoa with reduced motility.
In order to detect the separated motile spermatozoa, spermatozoa detection means such as a spermatozoa detection filter may be provided at a sample outlet side of the sample separation filter, and spaced therefrom. The detection means may be integral with the apparatus, or it may be provided as a separate component thereof for inserting into the apparatus before, during or after placing the filter in contact with the liquid medium. The spermatozoa detection filter may have similar characteristics to the sample separation filter.
The filters may have a thickness of 100-2000 xcexcm, preferably 200-1000 xcexcm, and more preferably 400-800 xcexcm. For example, the filters may have a thickness of about 600 xcexcm. The minimum particle retention size of the filters may be 5-100 xcexcm, preferably 8-60 xcexcm, and more preferably 10-40 xcexcm. The filters may be fibrous, for example made of glass wool or polypropylene, or they may have a gel or a foam construction. For gel or foam constructions that are not self supporting, an underlying grid lattice or other support may be provided. A particularly preferred filter is a glass fibre filter, which may include a binder such as an acrylic ester. It will be appreciated that the use of a gel as the filter can avoid the need to supply liquid to its opposed surface, because the gel itself acts as suitable means for achieving this. Preferred gels are hyaluronic acid and methylcellulose.
A reagent or a combination of reagents may be located in the spermatozoa detection means which are directly or indirectly capable of generating a visual signal on interaction with spermatozoa. These reagent or combination of reagents may include antibodies that detect an antigen present on spermatozoa and/or may be capable of binding spermatozoa. Spermatozoa, when immobilised by such antibodies, could be visually detected using a visually detectable reagent which binds to spermatozoa. Antibodies to CD59, as discussed in WO99/66331, the complete disclosure of which are incorporated herein by reference, have been found to be suitable for this purpose.
A spermatozoa chemoattractant, such as follicular fluid as identified in WO99/66331, may be located in the spermatozoa detection means. Such spermatozoa chemoattractants are preferably located in a portion of the spermatozoa detection means distal from the sample separation filter.
A pick-up zone may be located either in the sample separation filter or the spermatozoa detection means, said pick-up zone comprising a reagent or combination of reagents which is/are capable of binding to spermatozoa and being transported therewith through the filter(s) to a detection area of the spermatozoa detection means. The reagent or combination of reagents of the pick-up zone may include antibodies that detect an antigen present on spermatozoa. These antibodies may be detectably labelled, for example with gold particles.
The antibodies that are located in the detection area of the spermatozoa detection means may recognise the same or a different spermatozoa antigen from those located in the pick-up zone.
The spermatozoa detection means may comprise a spermatozoa acrosome-lysing reagent and a means for detecting pH change. The spermatozoa acrosome-lysing reagent is typically a lysis buffer, and may comprise Proteinase K or the calcium ionophore A24297. The means for detecting pH change could be a pH sensitive probe or a pH indicator reagent capable of visually detecting a pH change, for example bromocresol purple.
The sample receiving surface of the sample separating filter may contain an enzymatic liquefaction agent, such as chymotrypsin, capable of causing semen liquefaction. The invention also provides a male fertility testing kit comprising an apparatus as described above, the kit further comprising a liquid release mechanism, wherein upon activation of the liquid release mechanism, liquid from a liquid supply is applied to said opposed surface of the sample separation filter to provide liquid communication with a spermatozoa detection means.
The spermatozoa detection means may be integral with the apparatus or it may be provided as a separate or separable component of the kit.
The kit may comprise an integral liquid supply, or an external liquid supply may be used. It will be appreciated that the liquid must be one in which motile spermatozoa remain motile for a sufficient period of time to migrate to the spermatozoa detection means, ie. the liquid is generally non-toxic to spermatozoa. However, the liquid may be such that is has a toxicity to spermatozoa that is sufficiently low that enough spermatozoa will successfully reach the detecting means. The liquid is preferably a buffer such as phosphate buffered saline (PBS) or Earle""s Balanced Salt Solution (EBSS), as described in WO99/66331.
It will be appreciated that, where a gel-based filter is used, the gel can act as its own liquid supply. It may be desirable, however, to supply liquid to the opposed surface of a gel-based filter, for instance to dilute a sample after separation through the filter.
The kit may comprise two or more separable components, for example the apparatus and a base unit for the apparatus to engage with. Application of the apparatus to the base unit may be adapted to activate the liquid release mechanism, thereby wetting the spermatozoa detection filter and sample separation means. Alternatively, a button release for the liquid may be provided. The wetting of the spermatozoa detecting filter may activate the detecting agents applied to the spermatozoa detecting means.
An overflow container for catching excess liquid applied to the apparatus upon activation of the liquid release mechanism may be provided.
The liquid supply may comprise a frangible compartment or portion, wherein the liquid release mechanism breaks the compartment to release liquid contained therein. The liquid thereby released may then be channelled by a ramp towards a well formed between the apparatus and a base portion of the kit. The compartment may be piercable by a liquid release mechanism in the form of a piercing mechanism, for example retained by a stop, the stop preventing the piercing mechanism from piercing the compartment until activated by a user.
The invention also provides a method of detecting the presence of motile sperm in a sample, comprising the steps of providing a filter having first and second surfaces, the filter permitting migration of the motile sperm therethrough when a liquid is applied to the second surface, applying the sample to the first surface, applying a liquid to the second surface, and detecting sperm that has migrated through the filter. The sample may be a mixture comprising motile and non-motile spermatozoa. The filter may be the filter contained within the apparatus or the kit as described above.